Novel glucose dehydrogenase and process for producing the dehydrogenase

ABSTRACT

A novel glucose dehydrogenase, which is an enzyme that has high substrate specificity, can be produced at a low cost, is not affected by oxygen dissolved in a measurement sample and, in particular, has superior thermal stability is obtained by culturing a microorganism belonging to the genus Burkhorderia and having glucose dehydrogenase producing ability in a medium and collecting glucose dehydrogenase from the medium and/or cells of the microorganism.

TECHNICAL FIELD

[0001] The present invention relates to a novel glucose dehydrogenaseand a method for producing the same, a DNA encoding the enzyme, arecombinant vector comprising the DNA encoding the enzyme, atransformant transformed with the recombinant vector, a novelmicroorganism producing the enzyme, a glucose sensor using an enzymeelectrode including the enzyme, the transformant or the microorganism,and a glucose assay kit.

BACKGROUND ART

[0002] Biosensors using an enzyme that specifically reacts with aparticular substrate are being actively developed in various industrialfields. As for a glucose sensor, which is one of the biosensors, inparticular, measurement methods and devices utilizing such methods arebeing actively developed mainly in medical fields.

[0003] The glucose sensor has a history of about 40 years since Clarkand Lyons first reported about a biosensor comprising glucose oxidaseand an oxygen electrode in combination in 1962 (L.c. Clark, J. andLyonas, C. “Electrode systems for continuous monitoring incardiovascular surgery.” Ann. n. y. Acad. Sci., 105: 20-45).

[0004] Thus, the adoption of glucose oxidase as an enzyme of the glucosesensor has a long history. This is because glucose oxidase shows highsubstrate specificity for glucose and superior thermal stability, thisenzyme can further be produced in a large scale, and its production costis lower than those of other enzymes.

[0005] The high substrate specificity means that this enzyme does notreact with a saccharide other than glucose, and this leads to anadvantage that accurate measurement can be achieved without error inmeasurement values.

[0006] Further, the superior thermal stability means that problemsconcerning denaturation of the enzyme and inactivation of its enzymaticactivity due to heat can be prevented, and this leads to an advantagethat accurate measurement can be performed over a long period of time.

[0007] However, although glucose oxidase has high substrate specificityand superior thermal stability and can be produced at a low cost, it hasa problem that the enzyme is affected by dissolved oxygen as describedbelow and this affects measurement results.

[0008] Meanwhile, in addition to glucose oxidase, a glucose sensorutilizing glucose dehydrogenase has also been developed. This enzyme isalso found in microorganisms.

[0009] For example, there are known glucose dehydrogenase derived fromBacillus bacteria (EC 1.1.1.47) and glucose dehydrogenase derived fromCryptococcus bacteria (EC 1.1.1.119).

[0010] The former glucose dehydrogenase (EC 1.1.1.47) is an enzyme thatcatalyzes a reaction of β-D-glucose+NAD(P)⁺D-δ-gluconolactone+NAD(P)H+H⁺, and the latter glucose dehydrogenase(EC1.1.1.119) is an enzyme that catalyzes a reaction of D-glucose+NADP⁺D-δ-gluconolactone+NADPH+H⁺. The aforementioned glucose dehydrogenasesderived from microorganisms are already marketed.

[0011] These glucose dehydrogenases have an advantage that they are notaffected by oxygen dissolved in a measurement sample. This leads to anadvantage that accurate measurement can be achieved without causingerrors in measurement results even when the measurement is performed inan environment in which the oxygen partial pressure is low, or ahigh-concentration sample requiring a large amount of oxygen is used forthe measurement.

[0012] However, although glucose dehydrogenase is not affected bydissolved oxygen, it has problems of poor thermal stability andsubstrate specificity poorer than that of glucose oxidase.

[0013] Therefore, an enzyme that overcomes disadvantages of both ofglucose oxidase and glucose dehydrogenase has been desired.

[0014] The inventors of the present invention reported results of theirstudies about glucose dehydrogenase using samples collected from soilnear hot springs in Sode K., Tsugawa W., Yamazaki T., Watanabe M.,Ogasawara N., and Tanaka M., Enzyme Microb. Technol., 19, 82-85 (1996);Yamazaki T., Tsugawa W. and Sode K., Appli. Biochemi. and Biotec.,77-79/0325 (1999); and Yamazaki T., Tsugawa W. and Sode K., Biotec.Lett., 21, 199-202 (1999).

[0015] However, a bacterial strain having oxygen-producing ability hadnot been identified at the stage of these studies.

DISCLOSURE OF THE INVENTION

[0016] An object of the present invention is to provide an enzyme thatovercomes the disadvantages of both of known glucose oxidase and glucosedehydrogenase, i.e., an enzyme that shows high substrate specificity andsuperior thermal stability, can be produced at a low cost and is notaffected by oxygen dissolved in a measurement sample.

[0017] Further, another object of the present invention is to provide amethod for producing the aforementioned enzyme, a protein utilizingcharacteristics of the enzyme and a novel microorganism producing theenzyme.

[0018] A further object of the present invention is to provide a DNAencoding the aforementioned enzyme, a recombinant vector containing theDNA encoding the enzyme and a transformant transformed with therecombinant vector.

[0019] A still further object of the present invention is to provide aglucose sensor using an enzyme electrode including the aforementionedenzyme, transformant or microorganism and a glucose assay kit includingthe aforementioned enzyme.

[0020] The inventors of the present invention successfully isolatedBurkhorderia cepacia producing an enzyme achieving the aforementionedobjects from soil near hot springs, and thus accomplished the presentinvention.

[0021] Thus, the present invention provides the followings.

[0022] (1) A method for producing glucose dehydrogenase comprising thesteps of culturing a microorganism belonging to the genus Burkhorderiaand having glucose dehydrogenase producing ability in a medium, andcollecting glucose dehydrogenase from the medium and/or cells of themicroorganism.

[0023] (2) The method for producing glucose dehydrogenase according to(1), wherein the microorganism is Burkhorderia cepacia.

[0024] (3) The method for producing glucose dehydrogenase according to(1) or (2), wherein the glucose dehydrogenase has the followingproperties:

[0025] (i) the enzyme has an action of catalyzing dehydrogenationreaction of glucose;

[0026] (ii) the enzyme consists of subunits showing a molecular weightof about 60 kDa and a molecular weight of about 43 kDa inSDS-polyacrylamide gel electrophoresis under a reducing condition;

[0027] (iii) the enzyme shows a molecular weight of about 380 kDa in gelfiltration chromatography using TSK Gel G3000SW (Tosoh Corporation); and

[0028] (iv) the enzyme shows an optimal reaction temperature around 45°C. (Tris-HCl buffer, pH 8.0).

[0029] (4) The method for producing glucose dehydrogenase according to(3), wherein the subunit showing a molecular weight of about 43 kDa isan electron-transferring protein.

[0030] (5) The method for producing glucose dehydrogenase according to(4), wherein the electron-transferring protein is cytochrome C.

[0031] (6) A glucose dehydrogenase, which can be produced by amicroorganism belonging to the genus Burkhorderia.

[0032] (7) The glucose dehydrogenase according to (6), wherein themicroorganism is Burkhorderia cepacia.

[0033] (8) The glucose dehydrogenase according to (6) or (7), whereinthe glucose dehydrogenase has the following properties:

[0034] (i) the enzyme has an action of catalyzing dehydrogenationreaction of glucose;

[0035] (ii) the enzyme consists of subunits showing a molecular weightof about 60 kDa and a molecular weight of about 43 kDa inSDS-polyacrylamide gel electrophoresis under a reducing condition;

[0036] (iii) the enzyme shows a molecular weight of about 380 kDa in gelfiltration chromatography using TSK Gel G3000SW (Tosoh Corporation); and

[0037] (iv) the enzyme shows an optimal reaction temperature around 45°C. (Tris-HCl buffer, pH 8.0).

[0038] (9) The glucose dehydrogenase according to (8), wherein thesubunit showing a molecular weight of about 43 kDa is anelectron-transferring protein.

[0039] (10) The glucose dehydrogenase according to (9), wherein theelectron-transferring protein is cytochrome C.

[0040] (11) The glucose dehydrogenase according to any one of (8) to(10), wherein the subunit showing a molecular weight of about 60 kDacomprises the amino acid sequence of the amino acid numbers 2 to 12 inSEQ ID NO: 3.

[0041] (12) The glucose dehydrogenase according to any one of (8) to(11), wherein the N-terminus of the subunit showing a molecular weightof 43 kDa has the amino acid sequence of SEQ ID NO: 5.

[0042] (13) The glucose dehydrogenase according to (11), wherein thesubunit showing a molecular weight of about 60 kDa is a protein definedin the following (A) or (B):

[0043] (A) a protein which has the amino acid sequence of SEQ ID NO: 3;

[0044] (B) a protein which has the amino acid sequence of SEQ ID NO: 3including substitution, deletion, insertion or addition of one orseveral amino acid residues and a glucose dehydrogenase activity.

[0045] (14) The glucose dehydrogenase according to (6), which showsactivity peaks around 45° C. and around 75° C.

[0046] (15) A cytochrome C, which is a subunit of the glucosedehydrogenase according to (10) and has the amino acid sequence of SEQID NO: 5.

[0047] (16) A DNA encoding a part of the cytochrome C according to (15)and having the nucleotide sequence of SEQ ID NO: 8.

[0048] (17) A DNA encoding a part of the cytochrome C according to (15)and having the nucleotide sequence of the nucleotide numbers 2386 to2467 in the nucleotide sequence of SEQ ID NO: 1.

[0049] (18) A DNA encoding a signal peptide of the cytochrome Caccording to (15) and comprising the nucleotide sequence of thenucleotide numbers 2386 to 2451 in the nucleotide sequence of SEQ ID NO:1.

[0050] (19) A peptide which is a signal peptide of cytochrome C and hasthe amino acid sequence of the amino acid numbers 1 to 22 in the aminoacid sequence of SEQ ID NO: 4.

[0051] (20) A protein having the following properties:

[0052] (i) the protein can constitute the glucose dehydrogenaseaccording to (6) as a subunit;

[0053] (ii) the protein has a glucose dehydrogenase activity;

[0054] (iii) the protein shows a molecular weight of about 60 kDa inSDS-polyacrylamide gel electrophoresis under a reducing condition; and

[0055] (iv) the protein shows an optimal reaction temperature around 75°C. (Tris-HCl buffer, pH 8.0).

[0056] (21) The protein according to (20), which comprises the aminoacid sequence of the amino acid numbers 2 to 12 in SEQ ID NO: 3.

[0057] (22) The glucose dehydrogenase according to (21), wherein theprotein is a protein defined in the following (A) or (B) defined in thefollowing (A) or (B):

[0058] (A) a protein which has the amino acid sequence of SEQ ID NO: 3;

[0059] (B) a protein which has the amino acid sequence of SEQ ID NO: 3including substitution, deletion, insertion or addition of one orseveral amino acid residues and a glucose dehydrogenase activity.

[0060] (23) A protein defined in the following (A) or (B):

[0061] (A) a protein which has the amino acid sequence of SEQ ID NO: 3;

[0062] (B) a protein which has the amino acid sequence of SEQ ID NO: 3including substitution, deletion, insertion or addition of one orseveral amino acid residues and a glucose dehydrogenase activity.

[0063] (24) A DNA encoding a protein defined in the following (A) or(B):

[0064] (A) a protein which has the amino acid sequence of SEQ ID NO: 3;

[0065] (B) a protein which has the amino acid sequence of SEQ ID NO: 3including substitution, deletion, insertion or addition of one orseveral amino acid residues and a glucose dehydrogenase activity.

[0066] (25) The DNA according to (24), which is a DNA defined in thefollowing (a) or (b):

[0067] (a) a DNA which comprises the nucleotide sequence of thenucleotide numbers 764 to 2380 in the nucleotide sequence of SEQ ID NO:1;

[0068] (b) a DNA which is hybridizable with a nucleotide sequencecomprising the sequence of the nucleotide numbers 764 to 2380 in SEQ IDNO: 1 or a probe that can be prepared from the sequence under astringent condition and encodes a protein having a glucose dehydrogenaseactivity.

[0069] (26) A recombinant vector comprising the DNA according to (24) or(25).

[0070] (27) The recombinant vector according to (26), which comprisesnucleotide sequences encoding the signal peptide according to (18) and aβ-subunit.

[0071] (28) A transformant transformed with the DNA according to (24) or(25) or the recombinant vector according to (26) or (27).

[0072] (29) A method for producing glucose dehydrogenase comprising thesteps of culturing the transformant according to (28) to produce glucosedehydrogenase as an expression product of the DNA, and collecting it.

[0073] (30) A Burkhorderia cepacia KS1 strain (FERM BP-7306).

[0074] (31) A glucose sensor using an enzyme electrode including theglucose dehydrogenase according to any one of (6) to (14), the proteinaccording to any one of (20) to (23), the transformant according to (27)or the strain according to (30).

[0075] (32) A glucose assay kit including the glucose dehydrogenaseaccording to any one of (6) to (14) or the protein according to any oneof (20) to (23).

[0076] (33) A protein having the amino acid sequence of SEQ ID NO: 2.

[0077] (34) A DNA encoding a protein having the amino acid sequence ofSEQ ID NO: 2.

[0078] (35) The DNA according to (34), which comprises the nucleotidesequence of the nucleotide numbers 258 to 761 in the nucleotide sequenceof SEQ ID NO: 1.

[0079] (36) A DNA comprising the DNA according to (34) or (35) and theDNA according to (24) or (25) in this order.

[0080] (37) The DNA according to (36), which comprises the nucleotidesequence of the nucleotide numbers 258 to 2380 in the nucleotidesequence of SEQ ID NO: 1.

[0081] (38) A recombinant vector comprising the DNA according to (36) or(37).

[0082] (39) The recombinant vector according to (38), which comprisesnucleotide sequences encoding the signal peptide according to (18) and aβ-subunit.

[0083] (40) A transformant transformed with the DNA according to (36) or(37) or the recombinant vector according to (38) or (39).

[0084] (41) A method for producing glucose dehydrogenase comprising thesteps of culturing the transformant according to (40) to produce glucosedehydrogenase as an expression substance of the DNA according to (36) or(37), and collecting it.

[0085] Hereafter, the present invention will be explained in detail.

[0086] <1> Novel Bacterial Strain Producing Glucose Dehydrogenase of thePresent Invention.

[0087] The enzyme of the present invention (hereinafter, also referredto as “the enzyme” or “GDH”) can be produced by a bacterium belonging tothe genus Burkhorderia. The Burkhorderia bacterium used for the presentinvention is not particularly limited so long as it is a Burkhorderiabacterium having ability to produce the enzyme. However, Burkhorderiacepacia, in particular, the Burkhorderia cepacia KS1 strain ispreferred. This bacterial strain is a novel bacterial strain isolated bythe inventors of the present invention from soil near hot springs asdescribed later in the examples and was identified as Burkhorderiacepacia based on its bacteriological properties. Conventionally, it hasbeen unknown that a microorganism belonging to the genus Burkhorderiacan produce glucose dehydrogenase. This bacterial strain was designatedas KS1 strain. This strain was deposited at International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology (Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan, postal code: 305-8566) on Sep. 25, 2000 and receiveda microorganism accession number of FERM BP-7306.

[0088] The inventors of the present invention obtained some Burkhorderiacepacia strains other than the Burkhorderia cepacia KS1 strain, whichwere deposited at Institute for Fermentation (Osaka, IFO) or JapanCollection of Microorganisms (JCM), the Institute of Physical andChemical Research, and measured their glucose dehydrogenase activities.As a result, they confirmed that all of these bacterial strains had theactivity.

[0089] <2> Glucose Dehydrogenase of the Present Invention

[0090] If a Burkhorderia bacterium having glucose dehydrogenaseproducing ability, for example, the Burkhorderia cepacia KS1 strain, iscultured in a nutrient medium used for usual culture of a microorganism,preferably a medium containing glucose or a substance containing glucosein order to increase the enzyme producing ability, the glucosedehydrogenase of the present invention is produced and accumulated in aculture product or cultured cells. Therefore, it can be collected by aknown method. The method for producing the enzyme will be specificallyexplained by exemplifying the Burkhaorderia cepacia KS1 strain. First,the Burkhorderia cepacia KS1 strain is cultured in a suitable nutrientmedium, for example, a medium containing suitable carbon source,nitrogen source, inorganic salts, glucose or substances containing theseand so forth to produce and accumulate the enzyme in the culture productor the cultured cells.

[0091] As the carbon sources, any substance that can be assimilated canbe used, and examples include, for example, D-glucose, L-arabinose,D-xylose, D-mannose, starch, various peptones and so forth. As thenitrogen sources, there can be used yeast extract, malt extract, variouspeptones, various meat extracts, corn steep liquor, amino acid solutionsand organic and inorganic nitrogen compounds such as ammonium salts orsubstances containing these. As the inorganic salts, there can be usedvarious phosphoric acid salts and salts of magnesium, potassium, sodium,calcium and so forth. Further, as required, various inorganic andorganic substances required for growth of the bacterium or production ofthe enzyme, for example, silicone oil, sesame oil, defoaming agents suchas various surfactants and vitamins can be added to the medium.

[0092] As for the culture method, although either liquid culture orsolid culture may be used, liquid culture is usually preferred.

[0093] The enzyme of the present invention can be obtained from themedium and/or the cells in the culture obtained as described above. Theenzyme existing in the cells can be obtained as a cell extract bydisrupting or lysing the cells.

[0094] The glucose dehydrogenase in the culture product or the cellextract can be purified by a suitable combination of chromatographytechniques using an ion exchanger, a gel filtration carrier, ahydrophobic carrier and so forth.

[0095] The activity of the enzyme can be measured by the same methods asknown methods for measurement of the glucose dehydrogenase activity.Specifically, the activity can be measured by, for example, the methoddescribed later in the examples.

[0096] Physicochemical properties of the novel glucose dehydrogenase ofthe present invention are as follows:

[0097] (i) the enzyme has an action of catalyzing dehydrogenationreaction of glucose;

[0098] (ii) the enzyme consists of subunits showing a molecular weightof about 60 kDa and a molecular weight of about 43 kDa inSDS-polyacrylamide gel electrophoresis under a reducing condition;

[0099] (iii) the enzyme shows a molecular weight of about 380 kDa in gelfiltration chromatography using TSK Gel G3000SW (Tosoh Corporation); and

[0100] (iv) the enzyme shows an optimal reaction temperature around 45°C. (Tris-HCl buffer, pH 8.0).

[0101] The glucose dehydrogenase shows an activity peak around 45° C.under the aforementioned condition, and also shows an activity peakaround 75° C. (refer to FIG. 3(a)). No GDH has been known which showsthe activity peak in two of temperature regions as described above.

[0102] The molecular weight and the optimal temperature can be measuredby the methods described later in the examples.

[0103] The aforementioned glucose dehydrogenase of the present inventionconsists of two of separate polypeptides, the α-subunit having amolecular weight of about 60 kDa and the β-subunit having a molecularweight of about 43 kDa (hereinafter, this glucose dehydrogenase is alsoreferred to as “multimer enzyme”). The inventors of the presentinvention further investigated these two of subunits in detail.

[0104] It was found that the β-subunit was cytochrome C (as shown laterin the examples). A protein containing only the α-subunit exhibits thefollowing physicochemical properties:

[0105] (i) the protein can constitute the glucose dehydrogenase as asubunit;

[0106] (ii) the protein has a glucose dehydrogenase activity;

[0107] (iii) the protein shows a molecular weight of about 60 kDa inSDS-polyacrylamide gel electrophoresis under a reducing condition; and

[0108] (iv) the protein shows an optimal reaction temperature around 75°C. (Tris-HCl buffer, pH 8.0).

[0109] The optimal temperature can be measured by the method describedlater in the examples.

[0110] Since this protein itself has the enzymatic activity, the proteinmay be optionally called as “peptide enzyme” or “enzyme” depending onthe content of the explanation.

[0111] As a specific embodiment of the peptide enzyme of the presentinvention, a protein having the amino acid sequence of SEQ ID NO: 3 canbe mentioned. Further, this peptide enzyme may be a protein having theamino acid sequence containing substitution, deletion, insertion oraddition of one or more amino acid residues in the amino acid sequenceof SEQ ID NO: 3 so long as it has the GDH activity. Although an aminoacid sequence that can be encoded by the nucleotide sequence of SEQ IDNO: 1 is shown as SEQ ID NO: 3, the methionine residue at the N-terminusmay be eliminated after translation.

[0112] Further, as a specific embodiment of the multimer enzyme of thepresent invention, there can be mentioned a multimer containing aprotein of which α-subunit has the amino acid sequence of SEQ ID NO: 3.Further, the aforementioned multimer enzyme may be a multimer containinga protein of which α-subunit has the amino acid sequence of SEQ ID NO: 3including substitution, deletion, insertion or addition of one or moreamino acid residues, so long as it has the GDH activity.

[0113] In the present invention, “one or more” means a number of 1 to10, preferably 1 to 5, particularly preferably 1 to 3.

[0114] The inventors of the present invention confirmed existence of aγ-subunit in addition to the aforementioned α-subunit and β-subunit.

[0115] In the examples described later, the γ-subunit was removed at thestage of purifying the enzyme of the present invention from a culturesupernatant or cell extract, and therefore the γ-subunit was notconfirmed in the purified enzyme. However, as shown in the examples,when the γ-subunit was expressed together with the α-subunit, a highenzymatic activity was obtained in comparison with the case where onlythe α-subunit was expressed. This suggested that the γ-subunit was aprotein involved in the production of the α-subunit in a microbial cellin some sort of way. Assuming that the specific activity of theα-subunit (enzymatic activity per protein) is the same in either case, alower enzymatic activity indicates a smaller amount of the α-subunit asan enzyme since the enzymatic activity reflects the amount of theenzyme. On the other hand, the produced α-subunit may be protected bythe γ-subunit in a certain manner, or although the α-subunit as aprotein is fully expressed, it cannot have the three-dimensionalstructure for exhibiting the enzymatic activity due to the absence ofγ-subunit, and thus the enzymatic activity may become low. In eithercase, a high enzymatic activity can be obtained when the γ-subunit isexpressed together with the α-subunit.

[0116] <3> DNA of the Present Invention

[0117] The DNA of the present invention can be obtained from amicroorganism containing the DNA of the present invention, for example,Burkhorderia cepacia. The DNA of the present invention was isolated fromchromosomal DNA of Burkhorderia cepacia in the process of accomplishingthe present invention. However, since its nucleotide sequence and theamino acid sequence encoded by this nucleotide sequence were elucidatedby the present invention, the DNA can also be obtained by chemicalsynthesis based on those sequences. Further, the DNA of the presentinvention can also be obtained from chromosomal DNA of Burkhorderiacepacia or the like by hybridization or PCR using an oligonucleotideprepared based on the aforementioned sequences as a probe or a primer.

[0118] In addition to a DNA which encodes a protein having the aminoacid sequence of SEQ ID NO: 3, the DNA of the present invention may be aDNA which encodes a protein having an amino acid sequence of SEQ ID NO:3 containing substitution, deletion, insertion or addition of one ormore amino acid residues in the amino acid sequence and has the GDHactivity.

[0119] As the DNA of the present invention, there can be specificallymentioned a DNA comprising the nucleotide sequence of the nucleotidenumbers 764 to 2380 in the nucleotide sequence of SEQ ID NO: 1. Thenucleotide sequence of the nucleotide numbers 764 to 2380 in thenucleotide sequence of SEQ ID NO: 1 encodes the α-subunit of GDH havingthe amino acid sequence of SEQ ID NO: 3.

[0120] Further, the DNA of the present invention may also be a DNA whichis hybridizable with the nucleotide sequence of the nucleotide numbers764 to 2380 in the nucleotide sequence of SEQ ID NO: 1 or a probe thatcan be prepared from the sequence under a stringent conditions andencodes a protein having the GDH activity.

[0121] It is estimated that the nucleotide sequence of the nucleotidenumbers 258 to 761 in the nucleotide sequence of SEQ ID NO: 1 encodesthe γ-subunit. The amino acid sequence is shown in SEQ ID NO: 2. It isconsidered that, since the structural gene of the γ-subunit is includedin a region upstream from that of the α-subunit, and thus the γ-subunitis expressed first and already exists as a protein upon the productionof the α-subunit by a microorganism, the α-subunit can be efficientlyproduced in the microorganism. Therefore, the DNA of the presentinvention may include a DNA encoding the amino acid sequence of SEQ IDNO: 2 in addition to the aforementioned DNA.

[0122] A DNA encoding a protein substantially identical to theaforementioned protein having the amino acid sequence of SEQ ID NO: 3can be obtained by, for example, a method such as the site-directedmutagenesis or mutagenesis treatment. The GDH activity of a proteinencoded by a DNA introduced with a mutation can be measured, forexample, as follows.

[0123] An enzyme sample and glucose as a substrate are added to 10 mMpotassium phosphate buffer (pH 7.0) containing 594 μM methylphenazinemethosulfate (mPMS) and 5.94 μM 2,6-dichlorophenol-indopheol (DCIP) andincubated at 37° C. Change in absorbance of the DCIP at 600 nm ismonitored by using a spectrophotometer, and the absorbance decreasingrate is measured as an enzymatic reaction rate.

[0124] Further, the nucleotide sequence consisting of the nucleotide ofthe nucleotide number 2386 and the sequence after the nucleotide of thenucleotide number 2386 in the nucleotide sequence of SEQ ID NO: 1 isestimated to encode the β-subunit. Further, the nucleotide sequence ofthe nucleotide numbers 2386 to 2451 is estimated to encode the signalpeptide of the β-subunit. An estimated amino acid sequence of thissignal peptide is the amino acid sequence of amino acid numbers 1 to 22in SEQ ID NO: 4. The signal peptide is a peptide necessary for a proteinsynthesized in ribosome to be secreted through the membrane and has beenfound to comprise 15 to 30 hydrophobic amino acid residues. Therefore,since the amount of proteins in the culture supernatant is increased dueto the existence of the signal peptide, this is a peptide effectivelyacting in a method of producing a protein.

[0125] Hereafter, an example of a method for obtaining the DNA of thepresent invention will be explained.

[0126] Chromosomal DNA is isolated from a microorganism such asBurkhorderia cepacia and purified, and the chromosomal DNA is cleaved byultrasonication, restriction enzyme treatment or the like and ligated toa linear expression vector and cyclized by using a DNA ligase or thelike to construct a recombinant vector. The obtained recombinant vectoris introduced into a host microorganism in which the vector isautonomously replicable, and the transformants are screened by using avector marker and expression of an enzymatic activity as indexes toobtain a microorganism harboring a recombinant vector containing a geneencoding GDH. The recombinant vector contained in the obtainedmicroorganism is expected to contain at least the nucleotide sequenceencoding the α-subunit. Further, if the cloned fragment has a sufficientsize, it is very likely that the nucleotide sequence encoding theγ-subunit is also contained.

[0127] Then, the microorganism having the recombinant vector can becultured, the recombinant vector can be isolated from the cells of thecultured microorganism and purified, and the gene encoding GDH can becollected from the expression vector. For example, chromosomal DNAserving as a gene donor is specifically collected, for example, asfollows.

[0128] The aforementioned gene donor microorganism can be cultured withstirring for 1 to 3 days, for example, and cells can be collected bycentrifugation from the obtained culture broth and then lysed to preparecell lysate containing the GDH gene. As the method for lysis of thecells, a treatment is performed by using a bacteriolytic enzyme such aslysozyme, and other enzymes such as protease and surfactants such assodium dodecylsulfate (SDS) are used in combination as required.Further, a physical cell disruption method such as freeze and thawing orFrench press treatment may also be employed in combination.

[0129] The DNA can be isolated and purified from the lysate obtained asdescribed above in a conventional manner, for example, by a suitablecombination of deproteinization by phenol treatment or proteasetreatment, ribonuclease treatment, alcohol precipitation and so forth.

[0130] The DNA isolated and purified from a microorganism can be cleavedby, for example, ultrasonication, restriction enzyme treatment or thelike. Preferably, a type-II restriction enzyme, which acts on a specificnucleotide sequence, is suitably used. The restriction enzyme used maygenerate an end matching a digested end of a vector, or the digested endmay be blunt-ended by using an arbitrary restriction enzyme and ligatedto the vector.

[0131] As the vector used for cloning, a phage that can autonomouslygrow in a host microorganism or a plasmid that is constructed for generecombination is suitable. Examples of the phage include, for example,when Escherichia coli is used as the host microorganism, Lambda gt10,Lambda gt11 and so forth. Further, examples of the plasmid include, forexample, when Escherichia coli is used as the host microorganism,pBR322, pUC18, pUC118, pUC19, pUC119, pTrc99A and pBluescript as well asSuperCosI, which is a cosmid, and so forth.

[0132] Upon the cloning, a vector fragment can be obtained by digestingthe aforementioned vector with a restriction enzyme used for thedigestion of a microbial DNA as the aforementioned donor of a geneencoding GDH. However, a restriction enzyme identical to the restrictionenzyme used for the digestion of the microbial DNA does not necessarilyneed to be used. The method for ligating the microbial DNA fragment andthe vector DNA fragment may be a known method using a DNA ligase. Forexample, an adhesive end of the microbial DNA fragment and an adhesiveend of the vector fragment are ligated, and then a recombinant vectorcontaining the microbial DNA fragment and the vector DNA fragment isproduced by using a suitable DNA ligase. If required, after theligation, the fragment can also be introduced into the hostmicroorganism to produce the recombinant vector by utilizing a DNAligase existing in the microorganism.

[0133] The host microorganism used for the cloning is not particularlylimited so long as the recombinant vector is stable and can autonomouslygrow in the host, and a foreign gene can be expressed in the host.Escherichia coli DH5α, XL-1 BlueMR and so forth can generally be used.

[0134] As the method for introducing the recombinant vector into thehost microorganism, for example, when the host microorganism isEscherichia coli, the competent cell method using calcium treatment,electroporation or the like can be used.

[0135] Whether the cloned fragment obtained by the aforementioned methodencodes GDH can be confirmed by decoding the nucleotide sequence of thefragment in a conventional manner.

[0136] The DNA of the present invention can be obtained by collecting arecombinant vector from the transformant obtained as described above.

[0137] GDH can be produced by culturing a transformant containing theDNA of the present invention or a recombinant vector containing the DNAto produce GDH as an expression product of the DNA and collecting itfrom the cells or culture broth. For this production, although the DNAof the present invention may be a DNA encoding the α-subunit, theexpression efficiency can be increased by further expressing theγ-subunit together with the α-subunit.

[0138] Examples of the microorganism in which GDH is produced includeenteric bacteria such as Escherichia coli, Gram-negative bacteria suchas those of the genera Pseudomonas and Gluconobacter, Gram-positivebacteria including Bacillus bacteria such as Bacillus subtilis, yeastssuch as Saccharomyces cerevisiae and filamentous fungi such asAspergillus niger. However, the microorganism is not limited to thesemicroorganisms, and any host microorganism suitable for production offoreign proteins can be used.

[0139] The GDH gene contained in the once selected recombinant vectorcontaining the GDH gene can be easily transferred into a recombinantvector that can be replicated in a microorganism by recovering a DNAwhich is the GDH gene from the recombinant vector containing the GDHgene by using a restriction enzyme or by PCR and ligating it to anothervector fragment. Further, the microorganism can be easily transformedwith these vectors, for example, by the competent cell method usingcalcium treatment for Escherichia bacteria, the protoplast method forBacillus bacteria, the KU or KUR method for yeasts, themicromanipulation method for filamentous fungi and so forth. Further,electroporation can also be widely used.

[0140] The host microorganism into which a target recombinant vector isintroduced can be selected by searching a microorganism thatsimultaneously expresses a drug resistance marker of the vectorcontaining the target DNA and the GDH activity. For example, amicroorganism growing in a selective medium based on the drug resistancemarker and producing GDH can be selected.

[0141] As for the culture method of the transformant, culture conditionscan be selected by considering nutritional and physiological propertiesof the host. In many cases, liquid culture is performed. It isindustrially advantageous to perform aerobic culture with stirring.

[0142] As nutrients of the medium, those usually used for culture ofmicroorganisms can be widely used. As carbon sources, any carboncompounds that can be assimilated can be used, and examples thereofinclude glucose, sucrose, lactose, maltose, lactose, molasses, pyruvicacid and so forth. Further, as nitrogen sources, any nitrogen compoundsthat can be utilized can be used, and examples thereof include peptone,meat extracts, yeast extract, casein hydrolysate, soybean meal alkalineextract and so forth. In addition, phosphoric acid salts, carbonic acidsalts, sulfuric acid salts, salts of magnesium, calcium, potassium,iron, manganese, zinc and so forth, particular amino acids, particularvitamins and so forth are used as required.

[0143] Although the culture temperature can be appropriately changed ina range in which bacteria grow and produce GDH, it is preferably about20° C. to 42° C. The culture time somewhat varies depending on theconditions. However, the culture can be completed at an appropriate timeestimated to give the maximum GDH level, and the culture time is usuallyabout 12 to 72 hours. Although pH of the medium can be appropriatelychanged in a range in which bacteria grow and produce GDH, it ispreferably in the range of about pH 6.0 to 9.0.

[0144] The culture broth containing cells producing GDH in the culturecan be collected and utilized as they are. However, when GDH exists inthe culture broth, the culture broth is usually separated into aGDH-containing solution and microorganism cells by filtration,centrifugation or the like in a conventional manner and then used. WhenGDH exists in the cells, the cells are collected from the obtainedculture by means of filtration, centrifugation or the like, and then thecells are disrupted by a mechanical method or an enzymatic method suchas use of lysozyme, and further added with a chelating agent such asEDTA and a surfactant to solubilize GDH, as required, to isolate andcollect GHD as an aqueous solution.

[0145] GDH can be precipitated from the GDH-containing solution obtainedas described above by, for example, vacuum concentration, membraneconcentration, salting out with ammonium sulfate, sodium sulfate or thelike, or a fractional precipitation with a hydrophilic organic solventsuch as methanol, ethanol and acetone. Further, heat treatment andisoelectric point treatment are also effective purification means. Then,GDH can be purified by a suitable combination of gel filtration using anadsorbent or gel filtration agent, absorption chromatography, ionexchange chromatography and affinity chromatography to obtain purifiedGHD.

[0146] A purified enzyme preparation can be obtained by isolation andpurification based on column chromatography. Although the purifiedenzyme preparation is preferably purified to such an extent that asingle band should be obtained in electrophoresis (SDS-PAGE), it maycontain the γ-subunit.

[0147] The purified enzyme obtained as described above can be made intopowder by, for example, lyophilization, vacuum drying, spray drying orthe like and distributed.

[0148] Further, the amino acid sequence of the β-subunit can also bedetermined in the same manner as in the determination of the amino acidsequence of the α-subunit described later in the examples, and a DNAencoding the β-subunit can be isolated based on the sequence. Further,the β-subunit can also be produced by using the obtained DNA. Further,the multimer enzyme can also be produced by using a DNA encoding theα-subunit and DNA encoding the β-subunit.

[0149] <4> Glucose Sensor of the Present Invention

[0150] The glucose sensor of the present invention is characterized byusing the enzyme of the present invention (the aforementioned multimerenzyme or peptide enzyme, or the aforementioned multimer enzyme orpeptide enzyme containing the γ-subunit), the transformant of thepresent invention, or the microorganism of the present invention(Burkhorderia cepacia KS1 strain) as an enzyme electrode. As theelectrode, a carbon electrode, gold electrode, platinum electrode or thelike can be used, and the enzyme of the present invention is immobilizedon this electrode. Examples of the method for immobilization include amethod of using a crosslinking reagent, a method of entrapping theenzyme in a polymer matrix, a method of covering the enzyme with adialysis membrane, methods of using a photocrosslinking polymer,conductive polymer, oxidation-reduction polymer or the like.Alternatively, the enzyme may be immobilized in a polymer or immobilizedon an electrode by adsorption together with an electronic mediator ofwhich typical examples are ferrocene and derivatives thereof, or thesemethods may be used in combination. Typically, the glucose dehydrogenaseof the present invention is immobilized on a carbon electrode by usingglutaraldehyde, and glutaraldehyde is blocked by a treatment with areagent having an amine group.

[0151] The glucose concentration can be measured as follows. A buffer isplaced in a constant temperature cell and added with a mediator, and aconstant temperature is maintained. As the mediator, potassiumferricyanide, phenazine methosulfate and for forth can be used. Anelectrode on which the enzyme of the present invention is immobilized isused as a working electrode, and a counter electrode (e.g., platinumelectrode) and a reference electrode (e.g., Ag/AgC electrode) are used.A constant voltage is applied to the carbon electrode, and after asteady-state current is obtained, a sample containing glucose is addedand the increase of the current is measured. The glucose concentrationin the sample can be calculated according to a calibration curveproduced by using glucose solutions having standard concentrations.

[0152] <5> Glucose Assay Kit of the Present Invention

[0153] The saccharide assay kit of the present invention ischaracterized by including the enzyme of the present invention (theaforementioned multimer enzyme or peptide enzyme, or the aforementionedmultimer enzyme or peptide enzyme containing the γ-subunit). The glucoseassay kit of the present invention includes the enzyme of the presentinvention in an amount sufficient for at least one assay. Typically, thekit includes, in addition to the enzyme of the present invention, abuffer, a mediator, standard solutions of glucose or the like forcreating a calibration curve, which are necessary for the assay, and aguideline for use. The enzyme of the present invention can be providedin various forms, for example, as a lyophilized reagent or a solution inan appropriate storage solution.

BRIEF DESCRIPTION OF THE DRAWINGS

[0154]FIG. 1 shows a molecular weight of the enzyme of the presentinvention determined by native PAGE electrophoresis.

[0155]FIG. 2 shows an electrophoretic photograph showing a molecularweight of the enzyme of the present invention based on SDS-PAGEelectrophoresis.

[0156]FIG. 3 shows the optimal reaction temperature (a) and thermalstability (b) of the enzyme of the present invention.

[0157]FIG. 4 shows the optimal reaction temperature (a) and thermalstability (b) of a peptide enzyme constituting only the α-subunit of theenzyme of the present invention.

[0158]FIG. 5 shows results of spectrophotometric analyses of the enzymeof the present invention in the absence or presence of glucose beforeheat treatment (a) and spectrophotometric analyses of the enzyme of thepresent invention in the absence or presence of glucose after heattreatment (b).

[0159]FIG. 6 shows responses of a glucose sensor using GDH obtained froma transformant to glucose at various temperatures.

BEST MODE FOR CARRYING OUT THE INVENTION

[0160] The present invention will be explained more specifically withreference to the following examples.

EXAMPLE 1 Acquisition of Bacterium having Glucose DehydrogenaseProducing Ability

[0161] [Screening]

[0162] The microorganism of the present invention was obtained bycollecting soil near various hot springs in Japan and selecting abacterium having a glucose dehydrogenase activity among bacteriautilizing glucose as a nutrient from the soil.

[0163] The results of investigation of morphological characteristics,growth characteristics and physiological characteristics of this strainare shown below. [Bacteriological characteristics] Gram stainingnegative Cell morphology rod-shaped With polar flagellum positiveMobility Number of fragments >5 Optimal growth temperature 45° C.Oxidase negative Catalase positive Production of acetoin negativeProduction of H₂S negative Production of indole negative Acid fromglucose positive Arginine dihydrolase negative Urease negativeβ-Glucosidase negative Protease negative β-Galactosidase positive Lysinecarboxylase negative Ornithine carboxylase negative Reduction of nitratepositive [Assimilation characteristics] Glycerol positive Erythritolnegative D-Arabinose negative L-Arabinose positive Ribose positiveD-Xylose positive L-Xylose negative Adonitol positive β-Methyl-xylosidenegative Galactose positive D-Glucose positive D-Fructose positiveD-Mannose positive L-Sorbose negative Rhamnose negative Dulcitolpositive Inositol positive Mannitol positive Sorbitol positiveα-Methyl-D-mannoside negative α-Methyl-D-glucoside negativeN-Acetyl-glucosamine positive Amygdaline negative Arbutin negativeEsculin negative Salicin negative Cellobiose negative Maltose negativeLactose negative Melibiose negative Sucrose negative Trehalose positiveInulin negative Melezitose negative D-Raffinose negative Amidon negativeGlycogen negative Xylitol positive β-Gentiobiose negative D-Turanosenegative D-Lyxose negative D-Tagatose negative D-Fucose negativeL-Fucose negative D-Arabitol positive L-Arabitol positive Gluconic acidpositive 2-Ketogluconic acid positive 5-Ketogluconic acid negativeCapric acid positive Adipic acid positive Malic acid positive Citricacid positive Phenyl acetate positive [Oxidation characteristics]Glycerol negative Erythritol negative D-Arabinose negative L-Arabinosepositive Ribose positive D-Xylose positive L-Xylose negative Adonitolpositive β-Methyl-xyloside negative Galactose positive D-Glucosepositive D-Fructose positive D-Mannose positive L-Sorbose negativeRhamnose negative Dulcitol positive Inositol positive Mannitol positiveSorbitol positive α-Methyl-D-mannoside negative α-Methyl-D-glucosidenegative N-Acetyl-glucosamine negative Amygdaline negative Arbutinnegative Esculin positive Salicin negative Cellobiose positive Maltosepositive Lactose positive Melibiose negative Sucrose negative Trehalosepositive Inulin negative Melezitose negative D-Raffinose negative Amidonnegative Glycogen negative Xylitol negative β-Gentiobiose positiveD-Turanose negative D-Lyxose negative D-Tagatose negative D-Fucosepositive L -Fucose negative D-Arabitol positive L-Arabitol positiveGluconic acid negative 2-Ketogluconic acid negative 5-Ketogluconic acidnegative

[0164] The taxonomical position of the KS1 strain having theaforementioned bacteriological characteristics was investigated withreference to the Bergey's Manual of Determinative Bacteriology, and thestrain was identified to belong to the genus Burkhorderia, and was abacterial strain of Burkhorderia cepacia.

[0165] The genus Burkhorderia was conventionally classified into thegenus Pseudomonas, but is separately classified as the genusBurkhorderia at present (Yabuuchi, E., Kosako, Y., Oyaizu, H., Yano, I.,Hotta, H., Hashimoto, Y., Ezaki, T. and Arakawa, M., Microbiol. Immunol.Vol. 36 (12): 121-1275 (1992); International Journal of SystematicBacteriology, April, 1993, pp.398-399).

[0166] Further, the inventors of the present invention obtained severalBurkhorderia cepacia strains other than the Burkhorderia cepacia KS1strain, which were deposited at the Institute for Fermentation, Osaka orthe Japan Collection of Microorganisms (JCM), Institute of Physical andChemical Research, and measured glucose dehydrogenase activities of thestrains, and they were confirmed to have the activity. The glucosedehydrogenase activity was measured by the method described later inExample 2. Relative activities of these strains based on the enzymaticactivity of a water-soluble fraction of the KS1 strain, which is takenas 100, are shown in Table 1. TABLE 1 Glucose dehydrogenase Bacterialactivity strain 70° C. 45° C. KS1 Water-soluble 100 100 fraction JCM5506Water-soluble 100 100 fraction Membrane 100 100 fraction JCM5507Water-soluble 100 100 fraction Membrane 100 100 fraction JCM2800Water-soluble 100 100 fraction JCM2801 Water-soluble 100 100 fractionIFO15124 Water-soluble 100 100 fraction IFO14595 Water-soluble 100 100fraction

Example 2 Extraction of Glucose Dehydrogenase

[0167] <1> Culture of Cells

[0168] As the culture conditions of the bacterium, usual aerobic cultureconditions were used. The cells were cultured at 34° C. for 8 hours in 7L of a medium containing the following ingredients per liter.Polypeptone 10 g Yeast extract 1 g NaCl 5 g KH₂PO₄ 2 g Glucose 5 g Einol(ABLE Co., Tokyo, Japan) 0.14 g Total volume including distilled water 1L Adjusted pH 7.2

[0169] In a volume of 7 L of the culture broth was centrifuged at9,000×g at 4° C. for 10 minutes to obtain about 60 g of cells.

[0170] <2> Preparation of Roughly Purified Fraction

[0171] In an amount of 60 g of the cells were dispersed in 10 mMpotassium phosphate buffer (pH 6.0), and a pressure difference of 1,500Kg/cm² was applied to the cells by using a French press (OtakeCorporation, Tokyo, Japan) to disrupt cell membranes. The cell extractwas centrifuged at 8000×g for 10 minutes to remove cellular solid.Further, the supernatant was subjected to ultracentrifugation at69,800×g at 4° C. for 90 minutes to obtain about 8 g of a membranefraction as precipitates.

[0172] <3> Purification of Enzyme

[0173] The membrane fraction was redispersed in 10 mM potassiumphosphate buffer (pH 6.0) containing 1% of Triton X-100 as a finalconcentration. Then, the dispersion was slowly stirred overnight at 4°C. After the dispersion was subjected to ultracentrifugation (69,800×g,4° C., 90 minutes), the solubilized membrane fraction was centrifugedagain at 4° C. for 15 minutes at 15,000×g to obtain a supernatant.

[0174] The solubilized membrane fraction was added with the same volumeof 10 mM potassium phosphate buffer (pH 8.0) containing 0.2% TritonX-100. The solution was dialyzed, and then applied to a DEAE-TOYOPEARLcolumn (22 mm ID×20 cm, Tosoh Corporation, Tokyo, Japan) equalized with10 mM potassium phosphate buffer (pH 8.0) containing 0.2% Triton X-100.Proteins were eluted with a linear gradient of 0 to 0.15 M NaCl in 10 mMpotassium phosphate buffer (pH 8.0). The flow rate was 5 ml/min. GDH waseluted at a NaCl concentration of about 75 mM. Fractions exhibiting theGDH activity were collected and dialyzed overnight against 10 mMpotassium phosphate buffer (pH 8.0, 4° C.) containing 0.2% Triton X-100.

[0175] Further, the dialyzed enzyme solution was applied to a DEAE-5PWcolumn (8.0 mm ID ×7.5 cm, Tosoh Corporation, Tokyo, Japan). This columnwas equilibrated beforehand with 10 mM potassium phosphate buffer (pH6.0) containing 0.2% Triton X-100. The proteins were eluted with alinear gradient of 0 to 100 mM NaCl in 10 mM potassium phosphate buffer(pH 8.0). The flow rate was 1 ml/min. Fractions exhibiting the GDHactivity were eluted at a NaCl concentration of about 20 mM. Thefractions having the GDH activity were collected and desalted overnightwith 10 mM potassium phosphate buffer (pH 8.0) containing 0.2% TritonX-100 to obtain the purified enzyme.

[0176] The GDH activity was measured according to the following methodthroughout this example and the following examples.

[0177] As electron acceptors, 2,6-dichlorophenolindophenol (DCIP) andphenazine methosulfate (PMS) were used. The reaction was allowed in apolyethylene tube at a predetermined temperature. In a volume of 5 μl ofthe enzyme solution was added to 20 μl of 25 mM Tris-HCl buffer (pH 8.0)containing 0.75 mM PMS and 0.75 mM DCIP. This mixture was left for 1minute beforehand at a constant temperature. The reaction was startedwith the addition of 1 μl of 2 M glucose (final concentration: 77 mM)and left at a constant temperature for 2 minutes. Subsequently, 100 μlof ice-cooled distilled water or 120 μl of 7.5 M urea was added to coolthe sample. A reduction reaction of the electron acceptors due to thedehydrogenation of glucose was monitored by using an ultra-micromeasurement cell (100 μl) and a spectrophotometer (UV160, ShimadzuCorporation, Kyoto, Japan) that enabled measurement using the cell. Thatis, decoloration with time due to the reduction of DCIP was measured at600 nm, which is the absorption wavelength of DCIP. The molar absorbancecoefficient of DCIP (22.23 mM×cm⁻¹) was used. One unit (U) of the enzymewas defined as the amount of oxidizing 1 μM of glucose per minute understandard test conditions. The protein concentration was measured by theLowry method.

Example 3

[0178] Native PAGE electrophoresis was performed for the purifiedenzyme. The electrophoresis was performed on 8 to 25% polyacrylamidegradient gel using a Tris-alanine buffer system containing 1% TritonX-100. The gel was stained with silver nitrate. As protein markers,thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa),aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa)and chymotrypsinogen A (25 kDa) were used.

[0179] Further, activity staining was performed for the native PAGE gelby incubating the gel in the following solution for 30 minutes. At GDHactivity sites, nitroblue tetrazolium was reduced and formazan wasproduced, resulting in development of dark purple color. 200 mM glucose0.1 mM nitroblue tetrazolium 0.3 mM phenazine methosulfate 20 mMTris-HCl buffer (pH 8.0)

[0180] From the results of the silver staining in the native PAGE, itwas estimated that the enzyme consisted of a single kind of enzyme andhad a molecular weight of about 400 kDa. Further, when the gel wasstained for the activity, the activity was observed at a site of thesame mobility as in the silver staining (See FIG. 1. In the figure, Lane1 shows the results of silver staining of marker proteins havingstandard molecular weights, Lane 2 shows the silver staining of theenzyme, and Lane 4 shows the staining for activity of the enzyme). Whenthe enzyme was heated at 70° C. for 30 minutes, the activityunexpectedly remained, and the enzyme was separated into proteins one ofwhich had the activity and showed a molecular weight of about 85 kDa(See FIG. 1. In the figure, Lane 3 shows the results of the silverstaining of the enzyme heated at 70° C. for 30 minutes, and Lane 5 showsthe staining for activity of the enzyme heated at 70° C. for 30minutes). These results suggest that the enzyme consists of subunits.

Example 4

[0181] The purified enzyme solution was subjected to SDS-PAGE. SDS-PAGEwas performed in 8 to 25% gradient polyacrylamide gel by using aTris-tricine buffer. Proteins in the gel were stained with silvernitrate. Separation and development were automatically performed byusing Phast System (Pharmacia). The molecular mass was determined basedon the relative migrations of the standard proteins. The enzyme wasseparated into proteins having molecular weights of about 60 kDa and 43kDa by SDS-PAGE (See FIG. 2. FIG. 2 is an electrophoretic photograph. Inthe figure, Lane 1 shows the results of the silver nitrate staining ofthe marker proteins having standard molecular weights, and Lane 2 showsthe results of the silver nitrate staining of the enzyme). Thus, it wassuggested that the α-subunit of 60 kDa and the β-subunit of 43 kDa werebound in the enzyme, and it was expected that an octamer was formed byfour each of these subunits bonding to each other.

[0182] The β-subunit, a protein of 43 kDa separated by SDS-PAGE, wastransferred onto a polyvinylidene fluoride membrane, and then the aminoacid sequence at the N-terminus of the β-subunit was determined by usingan amino acid sequencer (PPSQ-10, Shimadzu Corporation). As a result, itwas found that the amino acid sequence at the N-terminus of the proteinconsisted of 16 residues of the amino acid sequence of SEQ ID NO: 5.

[0183] Further, the results obtained with the enzyme subjected to a heattreatment at 70° C. for 30 minutes are shown as Lane 3 in FIG. 2. Basedon this result of SDS-PAGE, it can be estimated that the enzyme waschanged into a single polypeptide having a molecular weight of 60 kDaafter the heat treatment.

Example 5

[0184] The enzyme was subjected to gel filtration chromatography. As thegel, TSK Gel G3000SW (Tosoh Corporation) was used, and the gel column(8.0 mm ID×30 cm Tosoh Corporation, Tokyo, Japan) was equilibrated witha solution containing 0.3 M NaCl and 0.1% Triton X100 in 10 mM potassiumphosphate buffer (pH 6.0). Fractions (125 μl) were collected. Sevenkinds of protein markers were used to determine the molecular weight ofthe purified enzyme. As the protein markers, thyroglobulin (669 kDa),ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serumalbumin (67 kDa), ovalbumin (43 kDa) and chymotrypsinogen A (25 kDa)were used.

[0185] It was confirmed that the molecular weight of the enzyme wasabout 380 kDa.

Example 6

[0186] The optimal temperature of the purified enzyme was examined.

[0187] The enzyme was incubated beforehand in Tris-HCl buffer (pH 8.0)at a predetermined temperature for 1 minute, and then the reaction wasstarted. The activity was measured at a predetermined reactiontemperature. The optimal temperature was observed around 45° C. (seeFIG. 3(a)). Further, a peak was also observed around 75° C., althoughthe activity was lower than the activity around 45° C.

[0188] Further, in order to examine thermal stability of the enzyme, theenzyme was left at each constant temperature for 30 minutes, and theresidual enzymatic activity was measured at 45° C. (see FIG. 3(b)).

Example 7

[0189] The optimal temperature and the thermal stability of the peptideenzyme constituting the single oligopeptide having a molecular weight of60 kDa obtained by heating the enzyme at 70° C. for 30 minutes wereexamined.

[0190] This peptide enzyme showed an optimal temperature higher thanthat of the unheated enzyme as well as thermal stability. There has beenno report about an enzyme having such temperature dependency.

[0191] The enzyme was incubated beforehand in Tris-HCl buffer (pH 8.0)at a predetermined temperature for 1 minute, and then the reaction wasstarted. The activity was measured at a predetermined reactiontemperature. The optimal temperature was observed around 75° C. (seeFIG. 4(a)).

[0192] Further, in order to examine thermal stability of the enzyme, theenzyme was left at each constant temperature for 30 minutes, and theresidual enzymatic activity was measured at 70° C. (see FIG. 4(b)).

Example 8

[0193] In order to investigate a role of each subunit,spectrophotometric analysis was performed for GDH before and after theheat treatment. FIGS. 5(a) and (b) show absorptions of oxidized andreduced GDHs before and after heat treatment (in the presence ofglucose). The absorption wavelength of the oxidized GDH before heattreatment, which was the original GDH, showed a characteristic peak at409 nm. Further, the peak shifted to 417 nm in the presence of glucose,and two more peaks were observed at 523 nm and 550 nm (FIG. 5(a)). Incontrast, the GDH after the heat treatment no longer showed thecharacteristic peak at 409 nm (FIG. 5(b)), and no significant differencewas observed between the oxidized and reduced GDHs.

[0194] The absorption wavelength of the oxidized GDH before heattreatment, which was the original GDH, was similar to the absorptionwavelength of alcohol dehydrogenase or aldehyde dehydrogenase comprisinga dehydrogenase cytochrome complex of Gluconobacter sp. or Acetobactersp. (refer to the following references: Adachi, O., Tayama, K.,Shinagawa, E., Matsushita, K. and Ameyama, M., Agr. Biol. Chem., 42,2045-2056 (1978); Adachi, O., Miyagawa, E., Matsushita, K. and Ameyama,M., Agr. Biol. Chem., 42, 2331-2340 (1978); Ameyama, M. and Adachi, O.,Methods Enzymol., 89, 450-457 (1982); Adachi, O., Tayama, K., Shinagawa,E., Matsushita, K. and Ameyama, M., Agr. Biol. Chem., 44, 503-515(1980); Ameyama, M. and Adachi, O., Methods Enzymol., 89, 491497(1982)).

[0195] The results indicated a possibility that the oligomer complex ofthe GDH contained cytochrome. Therefore, it can be considered that theobserved wavelength similar to that of cytochrome C is attributable tothe β-subunit and was lost during the heat treatment, and thus theβ-subunit consists of cytochrome C.

Example 9

[0196] A band containing the β-subunit obtained by the electrophoresisin Example 4 was excised, and the amino acid sequence was analyzed byusing a peptide sequencer (PPSQ-10, Shimadzu Corporation). As a result,the N-terminus amino acid sequence consisting of 16 residues shown inSEQ ID NO: 5 could be obtained.

[0197] It was attempted to amplify a gene region encoding theaforementioned N-terminus amino acid sequence of 16 residues by PCRbased on the peptide sequence. That is, two of PCR primers weredesigned, which had a nucleotide sequence on the forward side (SEQ IDNO: 6) corresponding to 5 residues at the N-terminus and a nucleotidesequence on the reverse side (SEQ ID NO: 7) corresponding to theantisense strand of 5 residues at the C-terminus in the peptide chain ofthe 16 residues. When PCR was performed in a conventional manner for theKS1 strain genome by using this pair of PCR primers, a gene fragment ofabout 50 bp was amplified. When the nucleotide sequence of this genefragment was determined in a conventional manner, a nucleotide sequenceof 58 nucleotides containing the pair of PCR primers were decoded. Amongthese nucleotides, 18 nucleotides excluding the PCR primers wereanalyzed, and a gene sequence corresponding to a region from Pro, whichwas the 6th residue from the N-terminus side of the aforementioned 16residues at the N-terminus of the β-subunit, to Arg, which was the 11thresidue, was found (SEQ ID NO: 8). Thus, it was found that the amplifiedgene fragment included the gene fragment of the β-subunit.

[0198] Further, it was also found that the β-subunit existed after 22amino acid residues following the α-subunit. This was based on a findingthat, since the amino acid sequence at the N-terminus of the purifiedβ-subunit determined in Example 4 matched 5 amino acid residuestranslated from the nucleotide sequence of the nucleotide numbers 2452to 2466 in SEQ ID NO: 1, these sequences are identical.

[0199] Furthermore, it is inferred that the nucleotide sequence of thenucleotide numbers 2386 to 2451 in SEQ ID NO: 1 is the signal peptide ofthe β-subunit. The amino acid sequence encoded by this nucleotidesequence corresponds to the amino acid numbers 1 to 22 in the amino acidsequence of SEQ ID NO: 4.

Example 10

[0200] The purified enzyme and a commercially available NAD coenzyme GDH(abbreviated as “NAD-GDH”) were added and mixed in 50 mM potassiumphosphate buffer (pH 7.5) containing 0.1% Triton X-100 and 1 mM CaCl₂ ata concentration of 100 U/L each. Each solution was placed in a hot tankat 60° C., and the residual activity was measured. TABLE 2 Residualrelative activity (%) Time (min) NAD-GDH The enzyme GDH 0 100 100 15 20100 30 5 100

[0201] It was confirmed that the enzyme had surprising thermal stabilityin comparison with that of the currently commercially available GDHenzyme. It was found that the enzyme was a novel enzyme that is totallydifferent from the commercially available NAD-GDH.

Example 10 Isolation of Gene Encoding α-Subunit of GDH

[0202] <1> Preparation of Chromosomal DNA from Burkhorderia cepacia KS1Strain

[0203] A chromosomal gene was prepared from the Burkhorderia cepacia KS1strain in a conventional manner. That is, the bacterial strain wasshaken overnight at 34° C. by using a TL liquid medium (10 g ofpolypeptone, 1 g of yeast extract, 5 g of NaCl, 2 g of KH₂PO₄, 5 g ofglucose in 1 L, pH 7.2). The grown cells were collected by using acentrifugal machine. The cells were suspended in a solution containing10 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% SDS and 100 μg/mlproteinase K and treated at 50° C. for 6 hours. This mixture was addedwith an equivalent volume of phenol-chloroform and stirred at roomtemperature for 10 minutes, and then the supernatant was collected byusing a centrifugal machine. The supernatant was added with sodiumacetate at a final concentration of 0.3 M and overlaid with two-foldvolume of ethanol to precipitate chromosomal DNA in the intermediatelayer. The DNA was taken up with a glass rod, washed with 70% ethanoland dissolved in an appropriate amount of TE buffer to obtain achromosomal DNA solution.

[0204] <2> Determination of N-Terminus Amino Acid Sequence of α-Subunitof GDH

[0205] GDH purified in the same manner as in Example 2 was concentratedby lyophilization and developed by SD-Selectrophoresis using 12.5%polyacrylamide to isolate the α-subunit. The α-subunit thus obtained wastransferred onto a polyvinylidene fluoride membrane, and then theN-terminus amino acid sequence was determined by using an amino acidsequencer (PPSQ-10, Shimadzu Corporation). As a result, it was foundthat the enzyme included a peptide sequence consisting of 11 residues ofthe amino acid numbers 2 to 12 in the amino acid sequence of SEQ ID NO:3.

[0206] <3> Cloning of Gene Encoding α-Subunit

[0207] In an amount of 1 μg of the DNA prepared in <1> was subjected tolimited digestion with a restriction enzyme Sau3AI and treated with calfintestinal alkaline phosphatase (CIAP). Separately, SuperCosI (obtainedfrom STRATAGENE), which is a cosmid, was treated with BamHI, and the DNAfragment obtained by the limited digestion of the chromosomal DNAfragment derived from the α-15 strain with Sau3AI was incorporated intoSuperCosI by using T4 DNA ligase. Escherichia coli XL-1 Blue MR(obtained from STRATAGENE) was transformed with the obtained recombinantDNA. Transformants were selected on an LB agar medium containing 10μg/ml neomycin and 25 μg/ml ampicillin based on neomycin resistance andampicillin resistance, which are antibiotic resistances of SuperCosI.The obtained transformants were cultured in the LB liquid medium. Thesetransformant cells were collected and suspended in a reagent formeasuring the GDH activity, and clones were selected by usingdehydrogenase activity for glucose as an index. As a result, one clonestrain showing the glucose dehydrogenase activity was obtained.

[0208] <4> Subcloning

[0209] DNA fragments containing the target gene were prepared from thecosmid, SuperCosI, containing the gene encoding the a-subunit obtainedin <3>. The inserted gene fragments were excised from the cosmid byusing a restriction enzyme NotI. These DNA fragments were treated with arestriction enzyme XbaI and incorporated into plasmid pUC18 digestedwith XbaI. The Escherichia coli DH5aMCR strain was transformed with theplasmid pUC18 containing each insert fragment, and colonies grown on anLB agar medium containing 50 μg/ml ampicillin were collected. Theobtained transformants were cultured in a liquid LB medium and examinedfor the GDH activity in the cells in the same manner as in <3>. As aresult, a strain showing the GDH activity was obtained from onetransformant. The plasmid was extracted from this transformant, and theinserted DNA fragment was analyzed. As a result, an insert fragment ofabout 8.8 kbp was confirmed. This plasmid was designated as pKS1.

[0210] <5> Determination of Nucleotide Sequence

[0211] The nucleotide sequence of the inserted DNA fragment in pKS1 wasdetermined according to the restriction enzyme analysis and aconventional method. As a result, the sequence of the DNA encoding theN-terminus amino acid sequence of the a-subunit found in <2> wasconfirmed in this inserted DNA fragment, and an open reading framecontaining this sequence was found. The determined nucleotide sequenceand the amino acid sequence that can be encoded by this nucleotidesequence are as shown in SEQ ID NOS: 1 and 3. The molecular weight of aprotein obtained from the amino acid sequence was 59,831 Da andsubstantially matched the molecular weight of 60 kDa obtained bySDS-PAGE of the α-subunit of the Burkhorderia cepacia KS1 strain.

[0212] Since the nucleotide sequence of the α-subunit was determined, avector was produced by using the aforementioned structural gene of theα-subunit, and a transformant was further produced with this vector.

[0213] First, a gene to be inserted into the vector was prepared asfollows.

[0214] Amplification was performed by PCR using a genome fragmentderived from the KS1 strain as a template so that a desired restrictionenzyme site should be included. The following pair of oligonucleotideprimers were used in PCR.

[0215] (Forward)

[0216] 5′-CCCAAGCTTGGGCCGATACCGATACGCA-3′ (SEQ ID NO: 9)

[0217] (Reverse)

[0218] 5′-GAGAAGCTTTCCGCACGGTCAGACTTCC-3′ (SEQ ID NO: 10)

[0219] The gene amplified by PCR was digested with a restriction enzymeHindIII and inserted into the expression vector pFLAG-CTS (SIGMA) at itscloning site, HindIII site. The obtained plasmid was designated aspFLAG-CTS/α.

[0220] The Escherichia coli DH5αMCR strain was transformed with theaforementioned plasmid pFLAG-CTS/α, and a colony grown on an LB agarmedium containing 50 μg/ml ampicillin was collected.

[0221] Further, when the open reading frame of the pKS1 insert fragmentwas searched in the upstream of the α-subunit, a structural gene of 507nucleotides encoding a polypeptide comprising 168 amino acid residuesshown in SEQ ID NO: 2 (nucleotide numbers 258 to 761 in SEQ ID NO: 1)was newly found. This structural gene was considered to encode theγ-subunit.

[0222] Since it was found that the region encoding the γ-subunit existedupstream from the coding region of the α-subunit, a recombinant vectorcontaining a gene having a polycistronic structure continuouslyincluding the γ-subunit and the α-subunit was produced, and atransformant introduced with this vector was constructed.

[0223] First, a gene to be inserted into the vector was prepared asfollows.

[0224] Amplification was performed by PCR using a genome fragment of theKS1 strain continuously including the structural gene of the γ-subunitand the structural gene of the α-subunit as a template so that a desiredrestriction enzyme site should be included. The following pair ofoligonucleotide primers were used for PCR.

[0225] (Forward)

[0226] 5′-CATGCCATGGCACACAACGACAACACT-3′ (SEQ ID NO: 11)

[0227] (Reverse)

[0228] 5′-CCCAAGCTTGGGTCAGACTTCCTTCTTCAGC-3′ (SEQ ID NO: 12)

[0229] The 5′ end and the 3′ end of the gene amplified by PCR weredigested with NcoI and HindIII, respectively, and the gene was insertedinto the vector pTrc99A (Pharmacia) at its cloning site, NcoI/HindIIIsite. The obtained plasmid was designated as pTrc99A/γ+α.

[0230] The Escherichia coli DH5αMCR strain was transformed with theaforementioned plasmid pTrc99A/γ+α, and a colony grown on an LB agarmedium containing 50 μg/ml ampicillin was collected.

Example 11 Production of α-Subunit of GDH by Recombinant Escherichiacoli

[0231] The α-subunit was produced by using the Escherichia coli DH5αMCRstrain transformed with each of the aforementioned plasmids pKS1,pFLAG-CTS/α and pTrc99A/γ+α. Each transformant was inoculated into 3 mlof LB medium containing 50 μg/ml ampicillin and cultured at 37° C. for12 hours, and cells were collected by using a centrifugal machine. Thecells were disrupted by using a French press (1500 kgf), and a membranefraction (10 mM potassium phosphate buffer, pH 6.0) was isolated byultracentrifugation (160,400×g, 4° C., 90 minutes).

Example 12 Assay of Glucose

[0232] First, the GDH activity in each of the aforementioned membranefractions was confirmed. Specifically, visual determination wasperformed by using a 10 mM potassium phosphate buffer (pH 7.0)containing 594 μM methylphenazine methosulfate (mPMS) and 5.94 μM2,6-dichlorophenol-indopheol (DCIP). The results are shown below. Thenumber of + represents the degree of color change from blue tocolorless. Membrane fraction of cultured transformant + transformed withpFLAG-CTS/α: Membrane fraction of cultured transformant ++ transformedwith pKS1: Membrane fraction of cultured transformant +++ transformedwith pTrc99A/γ + α:

[0233] The GDH activity of the membrane fraction of the culturedtransformant transformed with pFLAG-CTS/α incorporated only with theα-subunit was the lowest, and the GDH activity of the membrane fractionof the cultured transformant transformed with pTrc99A/γ+α, with which avector was efficiently constructed, was the highest.

[0234] Although the α-subunit was expressed even in the transformanttransformed with a vector using only the structural gene of theα-subunit, the α-subunit could be efficiently obtained by using a vectorcontaining the structural gene of the γ-subunit and the structural geneof the α-subunit in combination.

[0235] Glucose was assayed by using the glucose dehydrogenase of thepresent invention. The enzymatic activity of the glucose dehydrogenase(α-subunit) of the present invention was measured by using glucose atvarious concentrations. The GDH activity was measured in 10 mM potassiumphosphate buffer (pH 7.0) containing 594 μM methylphenazine methosulfate(mPMS) and 5.94 μM 2,6-dichlorophenol-indopheol (DCIP). An enzyme sampleand glucose as a substrate were added and incubated at 37° C., andchange in the absorbance of DCIP at 600 nm was monitored by using aspectrophotometer. The absorbance decreasing rate was measured as anenzymatic reaction rate. Glucose could be quantified in the range of0.01 to 1.0 mM by using the GDH of the present invention.

Example 13 Preparation and Evaluation of Glucose Sensor

[0236] The glucose dehydrogenase (25 units) of the present inventionobtained in Example 2 was added with 20 mg of carbon paste andlyophilized. These were sufficiently mixed, applied only on a surface ofa carbon paste electrode already filled with about 40 mg of carbon pasteand polished on a filter paper. This electrode was treated in 10 mM MOPSbuffer (pH 7.0) containing 1% glutaraldehyde at room temperature for 30minutes and then treated in 10 mM MOPS buffer (pH 7.0) containing 20 mMlysine at room temperature for 20 minutes to block glutaraldehyde. Thiselectrode was equilibrated in 10 mM MOPS buffer (pH 7.0) at roomtemperature for 1 hour or longer. The electrode was stored at 4° C.

[0237] By using the aforementioned electrode as a working electrode, anAg/AgCl electrode as a reference electrode and a Pt electrode as acounter electrode, a response current value was measured upon additionof glucose. The 10 mM potassium phosphate buffer containing 1 mMmethoxy-PMS was used as the reaction solution, and a potential of 100 mVwas applied for the measurement.

[0238] Glucose concentration was measured by using the produced enzymesensor. Glucose could be quantified in the range of 0.05 to 5.0 mM byusing the enzyme sensor on which the glucose dehydrogenase of thepresent invention was immobilized (FIG. 6).

Example 14 Preparation and Evaluation of Glucose Sensor by GDH Obtainedfrom Transformant

[0239] In an amount of 10 U of the α-subunit (249 U/mg protein) of thepresent invention obtained in Example 12 was added with 50 mg of carbonpaste and lyophilized. These were sufficiently mixed, applied only on asurface of a carbon paste electrode already filled with about 40 mg ofcarbon paste and polished on a filter paper. This electrode was treatedin a 10 mM MOPS buffer (pH 7.0) containing 1% glutaraldehyde at roomtemperature for 30 minutes and then treated in 10 mM MOPS buffer (pH7.0) containing 20 mM lysine at room temperature for 20 minutes to blockglutaraldehyde. This electrode was equilibrated in 10 mM MOPS buffer (pH7.0) at room temperature for 1 hour or longer. The electrode was storedat 4° C.

[0240] By using the aforementioned electrode as a working electrode, anAg/AgCl electrode as a reference electrode and a Pt electrode as acounter electrode, a response current value was measured upon additionof glucose. The 10 mM potassium phosphate buffer containing 1 mMmethoxy-PMS was used as the reaction solution, and the measurement wasperformed for glucose aqueous solutions of various concentrations at 25°C. and 40° C. with applying a potential of 100 mV.

[0241] It was confirmed that, when the glucose concentration wasmeasured by using the produced enzyme sensor, a current corresponding toeach concentration was obtained.

INDUSTRIAL APPLICABILITY

[0242] According to the present invention, an enzyme that has highsubstrate specificity, can be produced at a low cost and is not affectedby oxygen dissolved in a measurement sample, in particular, novelglucose dehydrogenase having superior thermal stability, and a methodfor producing the enzyme could be provided. Further, a novel bacterialstrain of Burkhorderia cepacia producing the enzyme was obtained. Aglucose sensor effective for measurement of glucose can also be providedby using an enzyme electrode containing the enzyme or the bacterialstrain.

[0243] Further, since the glucose dehydrogenase gene, a peptide thatenables efficient expression of the gene and a DNA encoding this peptidewere found by the present invention, a large amount of GDH can beprepared by using recombinant DNA techniques based on the gene.

1 12 1 2467 DNA Burkhorderia cepacia CDS (258)..(761) CDS (764)..(2380)CDS (2386)..(2466) 1 aagctttctg tttgattgca cgcgattcta accgagcgtctgtgaggcgg aacgcgacat 60 gcttcgtgtc gcacacgtgt cgcgccgacg acacaaaaatgcagcgaaat ggctgatcgt 120 tacgaatggc tgacacattg aatggactat aaaaccattgtccgttccgg aatgtgcgcg 180 tacatttcag gtccgcgccg atttttgaga aatatcaagcgtggttttcc cgaatccggt 240 gttcgagaga aggaaac atg cac aac gac aac act ccccac tcg cgt cgc 290 Met His Asn Asp Asn Thr Pro His Ser Arg Arg 1 5 10cac ggc gac gca gcc gca tca ggc atc acg cgg cgt caa tgg ttg caa 338 HisGly Asp Ala Ala Ala Ser Gly Ile Thr Arg Arg Gln Trp Leu Gln 15 20 25 ggcgcg ctg gcg ctg acc gca gcg ggc ctc acg ggt tcg ctg aca ttg 386 Gly AlaLeu Ala Leu Thr Ala Ala Gly Leu Thr Gly Ser Leu Thr Leu 30 35 40 cgg gcgctt gca gac aac ccc ggc act gcg ccg ctc gat acg ttc atg 434 Arg Ala LeuAla Asp Asn Pro Gly Thr Ala Pro Leu Asp Thr Phe Met 45 50 55 acg ctt tccgaa tcg ctg acc ggc aag aaa ggg ctc agc cgc gtg atc 482 Thr Leu Ser GluSer Leu Thr Gly Lys Lys Gly Leu Ser Arg Val Ile 60 65 70 75 ggc gag cgcctg ctg cag gcg ctg cag aag ggc tcg ttc aag acg gcc 530 Gly Glu Arg LeuLeu Gln Ala Leu Gln Lys Gly Ser Phe Lys Thr Ala 80 85 90 gac agc ctg ccgcag ctc gcc ggc gcg ctc gcg tcc ggt tcg ctg acg 578 Asp Ser Leu Pro GlnLeu Ala Gly Ala Leu Ala Ser Gly Ser Leu Thr 95 100 105 cct gaa cag gaatcg ctc gca ctg acg atc ctc gag gcc tgg tat ctc 626 Pro Glu Gln Glu SerLeu Ala Leu Thr Ile Leu Glu Ala Trp Tyr Leu 110 115 120 ggc atc gtc gacaac gtc gtg att acg tac gag gaa gca tta atg ttc 674 Gly Ile Val Asp AsnVal Val Ile Thr Tyr Glu Glu Ala Leu Met Phe 125 130 135 ggc gtc gtg tccgat acg ctc gtg atc cgt tcg tat tgc ccc aac aaa 722 Gly Val Val Ser AspThr Leu Val Ile Arg Ser Tyr Cys Pro Asn Lys 140 145 150 155 ccc ggc ttctgg gcc gac aaa ccg atc gag agg caa gcc tg atg gcc 769 Pro Gly Phe TrpAla Asp Lys Pro Ile Glu Arg Gln Ala Met Ala 160 165 1 gat acc gat acgcaa aag gcc gac gtc gtc gtc gtt gga tcg ggt gtc 817 Asp Thr Asp Thr GlnLys Ala Asp Val Val Val Val Gly Ser Gly Val 5 10 15 gcg ggc gcg atc gtcgcg cat cag ctc gcg atg gcg ggc aag gcg gtg 865 Ala Gly Ala Ile Val AlaHis Gln Leu Ala Met Ala Gly Lys Ala Val 20 25 30 atc ctg ctc gaa gcg ggcccg cgc atg ccg cgc tgg gaa atc gtc gag 913 Ile Leu Leu Glu Ala Gly ProArg Met Pro Arg Trp Glu Ile Val Glu 35 40 45 50 cgc ttc cgc aat cag cccgac aag atg gac ttc atg gcg ccg tac ccg 961 Arg Phe Arg Asn Gln Pro AspLys Met Asp Phe Met Ala Pro Tyr Pro 55 60 65 tcg agc ccc tgg gcg ccg catccc gag tac ggc ccg ccg aac gac tac 1009 Ser Ser Pro Trp Ala Pro His ProGlu Tyr Gly Pro Pro Asn Asp Tyr 70 75 80 ctg atc ctg aag ggc gag cac aagttc aac tcg cag tac atc cgc gcg 1057 Leu Ile Leu Lys Gly Glu His Lys PheAsn Ser Gln Tyr Ile Arg Ala 85 90 95 gtg ggc ggc acg acg tgg cac tgg gccgcg tcg gcg tgg cgc ttc att 1105 Val Gly Gly Thr Thr Trp His Trp Ala AlaSer Ala Trp Arg Phe Ile 100 105 110 ccg aac gac ttc aag atg aag agc gtgtac ggc gtc ggc cgc gac tgg 1153 Pro Asn Asp Phe Lys Met Lys Ser Val TyrGly Val Gly Arg Asp Trp 115 120 125 130 ccg atc cag tac gac gat ctc gagccg tac tat cag cgc gcg gag gaa 1201 Pro Ile Gln Tyr Asp Asp Leu Glu ProTyr Tyr Gln Arg Ala Glu Glu 135 140 145 gag ctc ggc gtg tgg ggc ccg ggcccc gag gaa gat ctg tac tcg ccg 1249 Glu Leu Gly Val Trp Gly Pro Gly ProGlu Glu Asp Leu Tyr Ser Pro 150 155 160 cgc aag cag ccg tat ccg atg ccgccg ctg ccg ttg tcg ttc aac gag 1297 Arg Lys Gln Pro Tyr Pro Met Pro ProLeu Pro Leu Ser Phe Asn Glu 165 170 175 cag acc atc aag acg gcg ctg aacaac tac gat ccg aag ttc cat gtc 1345 Gln Thr Ile Lys Thr Ala Leu Asn AsnTyr Asp Pro Lys Phe His Val 180 185 190 gtg acc gag ccg gtc gcg cgc aacagc cgc ccg tac gac ggc cgc ccg 1393 Val Thr Glu Pro Val Ala Arg Asn SerArg Pro Tyr Asp Gly Arg Pro 195 200 205 210 act tgt tgc ggc aac aac aactgc atg ccg atc tgc ccg atc ggc gcg 1441 Thr Cys Cys Gly Asn Asn Asn CysMet Pro Ile Cys Pro Ile Gly Ala 215 220 225 atg tac aac ggc atc gtg cacgtc gag aag gcc gaa cgc gcc ggc gcg 1489 Met Tyr Asn Gly Ile Val His ValGlu Lys Ala Glu Arg Ala Gly Ala 230 235 240 aag ctg atc gag aac gcg gtcgtc tac aag ctc gag acg ggc ccg gac 1537 Lys Leu Ile Glu Asn Ala Val ValTyr Lys Leu Glu Thr Gly Pro Asp 245 250 255 aag cgc atc gtc gcg gcg ctctac aag gac aag acg ggc gcc gag cat 1585 Lys Arg Ile Val Ala Ala Leu TyrLys Asp Lys Thr Gly Ala Glu His 260 265 270 cgc gtc gaa ggc aag tat ttcgtg ctc gcc gcg aac ggc atc gag acg 1633 Arg Val Glu Gly Lys Tyr Phe ValLeu Ala Ala Asn Gly Ile Glu Thr 275 280 285 290 ccg aag atc ctg ctg atgtcc gcg aac cgc gat ttc ccg aac ggt gtc 1681 Pro Lys Ile Leu Leu Met SerAla Asn Arg Asp Phe Pro Asn Gly Val 295 300 305 gcg aac agc tcg gac atggtc ggc cgc aac ctg atg gac cat ccg ggc 1729 Ala Asn Ser Ser Asp Met ValGly Arg Asn Leu Met Asp His Pro Gly 310 315 320 acc ggc gtg tcg ttc tatgcg agc gag aag ctg tgg ccg ggc cgc ggc 1777 Thr Gly Val Ser Phe Tyr AlaSer Glu Lys Leu Trp Pro Gly Arg Gly 325 330 335 ccg cag gag atg acg tcgctg atc ggt ttc cgc gac ggt ccg ttc cgc 1825 Pro Gln Glu Met Thr Ser LeuIle Gly Phe Arg Asp Gly Pro Phe Arg 340 345 350 gcg acc gaa gcg gcg aagaag atc cac ctg tcg aac ctg tcg cgc atc 1873 Ala Thr Glu Ala Ala Lys LysIle His Leu Ser Asn Leu Ser Arg Ile 355 360 365 370 gac cag gag acg cagaag atc ttc aag gcc ggc aag ctg atg aag ccc 1921 Asp Gln Glu Thr Gln LysIle Phe Lys Ala Gly Lys Leu Met Lys Pro 375 380 385 gac gag ctc gac gcgcag atc cgc gac cgt tcc gca cgc tac gtg cag 1969 Asp Glu Leu Asp Ala GlnIle Arg Asp Arg Ser Ala Arg Tyr Val Gln 390 395 400 ttc gac tgc ttc cacgaa atc ctg ccg caa ccc gag aac cgc atc gtg 2017 Phe Asp Cys Phe His GluIle Leu Pro Gln Pro Glu Asn Arg Ile Val 405 410 415 ccg agc aag acg gcgacc gat gcg atc ggc att ccg cgc ccc gag atc 2065 Pro Ser Lys Thr Ala ThrAsp Ala Ile Gly Ile Pro Arg Pro Glu Ile 420 425 430 acg tat gcg atc gacgac tac gtg aag cgc ggc gcc gcg cat acg cgc 2113 Thr Tyr Ala Ile Asp AspTyr Val Lys Arg Gly Ala Ala His Thr Arg 435 440 445 450 gag gtc tac gcgacc gcc gcg aag gtg ctc ggc ggc acg gac gtc gtg 2161 Glu Val Tyr Ala ThrAla Ala Lys Val Leu Gly Gly Thr Asp Val Val 455 460 465 ttc aac gac gaattc gcg ccg aac aat cac atc acg ggc tcg acg atc 2209 Phe Asn Asp Glu PheAla Pro Asn Asn His Ile Thr Gly Ser Thr Ile 470 475 480 atg ggc gcc gatgcg cgc gac tcc gtc gtc gac aag gac tgc cgc acg 2257 Met Gly Ala Asp AlaArg Asp Ser Val Val Asp Lys Asp Cys Arg Thr 485 490 495 ttc gac cat ccgaac ctg ttc att tcg agc agc gcg acg atg ccg acc 2305 Phe Asp His Pro AsnLeu Phe Ile Ser Ser Ser Ala Thr Met Pro Thr 500 505 510 gtc ggt acc gtaaac gtg acg ctg acg atc gcc gcg ctc gcg ctg cgg 2353 Val Gly Thr Val AsnVal Thr Leu Thr Ile Ala Ala Leu Ala Leu Arg 515 520 525 530 atg tcg gacacg ctg aag aag gaa gtc tgacc gtg cgg aaa tct act ctc 2403 Met Ser AspThr Leu Lys Lys Glu Val Val Arg Lys Ser Thr Leu 535 1 5 act ttc ctc atcgcc ggc tgc ctc gcg ttg ccg ggc ttc gcg cgc gcg 2451 Thr Phe Leu Ile AlaGly Cys Leu Ala Leu Pro Gly Phe Ala Arg Ala 10 15 20 gcc gat gcg gcc gatc 2467 Ala Asp Ala Ala Asp 25 2 168 PRT Burkhorderia cepacia 2 Met HisAsn Asp Asn Thr Pro His Ser Arg Arg His Gly Asp Ala Ala 1 5 10 15 AlaSer Gly Ile Thr Arg Arg Gln Trp Leu Gln Gly Ala Leu Ala Leu 20 25 30 ThrAla Ala Gly Leu Thr Gly Ser Leu Thr Leu Arg Ala Leu Ala Asp 35 40 45 AsnPro Gly Thr Ala Pro Leu Asp Thr Phe Met Thr Leu Ser Glu Ser 50 55 60 LeuThr Gly Lys Lys Gly Leu Ser Arg Val Ile Gly Glu Arg Leu Leu 65 70 75 80Gln Ala Leu Gln Lys Gly Ser Phe Lys Thr Ala Asp Ser Leu Pro Gln 85 90 95Leu Ala Gly Ala Leu Ala Ser Gly Ser Leu Thr Pro Glu Gln Glu Ser 100 105110 Leu Ala Leu Thr Ile Leu Glu Ala Trp Tyr Leu Gly Ile Val Asp Asn 115120 125 Val Val Ile Thr Tyr Glu Glu Ala Leu Met Phe Gly Val Val Ser Asp130 135 140 Thr Leu Val Ile Arg Ser Tyr Cys Pro Asn Lys Pro Gly Phe TrpAla 145 150 155 160 Asp Lys Pro Ile Glu Arg Gln Ala 165 3 539 PRTBurkhorderia cepacia 3 Met Ala Asp Thr Asp Thr Gln Lys Ala Asp Val ValVal Val Gly Ser 1 5 10 15 Gly Val Ala Gly Ala Ile Val Ala His Gln LeuAla Met Ala Gly Lys 20 25 30 Ala Val Ile Leu Leu Glu Ala Gly Pro Arg MetPro Arg Trp Glu Ile 35 40 45 Val Glu Arg Phe Arg Asn Gln Pro Asp Lys MetAsp Phe Met Ala Pro 50 55 60 Tyr Pro Ser Ser Pro Trp Ala Pro His Pro GluTyr Gly Pro Pro Asn 65 70 75 80 Asp Tyr Leu Ile Leu Lys Gly Glu His LysPhe Asn Ser Gln Tyr Ile 85 90 95 Arg Ala Val Gly Gly Thr Thr Trp His TrpAla Ala Ser Ala Trp Arg 100 105 110 Phe Ile Pro Asn Asp Phe Lys Met LysSer Val Tyr Gly Val Gly Arg 115 120 125 Asp Trp Pro Ile Gln Tyr Asp AspLeu Glu Pro Tyr Tyr Gln Arg Ala 130 135 140 Glu Glu Glu Leu Gly Val TrpGly Pro Gly Pro Glu Glu Asp Leu Tyr 145 150 155 160 Ser Pro Arg Lys GlnPro Tyr Pro Met Pro Pro Leu Pro Leu Ser Phe 165 170 175 Asn Glu Gln ThrIle Lys Thr Ala Leu Asn Asn Tyr Asp Pro Lys Phe 180 185 190 His Val ValThr Glu Pro Val Ala Arg Asn Ser Arg Pro Tyr Asp Gly 195 200 205 Arg ProThr Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys Pro Ile 210 215 220 GlyAla Met Tyr Asn Gly Ile Val His Val Glu Lys Ala Glu Arg Ala 225 230 235240 Gly Ala Lys Leu Ile Glu Asn Ala Val Val Tyr Lys Leu Glu Thr Gly 245250 255 Pro Asp Lys Arg Ile Val Ala Ala Leu Tyr Lys Asp Lys Thr Gly Ala260 265 270 Glu His Arg Val Glu Gly Lys Tyr Phe Val Leu Ala Ala Asn GlyIle 275 280 285 Glu Thr Pro Lys Ile Leu Leu Met Ser Ala Asn Arg Asp PhePro Asn 290 295 300 Gly Val Ala Asn Ser Ser Asp Met Val Gly Arg Asn LeuMet Asp His 305 310 315 320 Pro Gly Thr Gly Val Ser Phe Tyr Ala Ser GluLys Leu Trp Pro Gly 325 330 335 Arg Gly Pro Gln Glu Met Thr Ser Leu IleGly Phe Arg Asp Gly Pro 340 345 350 Phe Arg Ala Thr Glu Ala Ala Lys LysIle His Leu Ser Asn Leu Ser 355 360 365 Arg Ile Asp Gln Glu Thr Gln LysIle Phe Lys Ala Gly Lys Leu Met 370 375 380 Lys Pro Asp Glu Leu Asp AlaGln Ile Arg Asp Arg Ser Ala Arg Tyr 385 390 395 400 Val Gln Phe Asp CysPhe His Glu Ile Leu Pro Gln Pro Glu Asn Arg 405 410 415 Ile Val Pro SerLys Thr Ala Thr Asp Ala Ile Gly Ile Pro Arg Pro 420 425 430 Glu Ile ThrTyr Ala Ile Asp Asp Tyr Val Lys Arg Gly Ala Ala His 435 440 445 Thr ArgGlu Val Tyr Ala Thr Ala Ala Lys Val Leu Gly Gly Thr Asp 450 455 460 ValVal Phe Asn Asp Glu Phe Ala Pro Asn Asn His Ile Thr Gly Ser 465 470 475480 Thr Ile Met Gly Ala Asp Ala Arg Asp Ser Val Val Asp Lys Asp Cys 485490 495 Arg Thr Phe Asp His Pro Asn Leu Phe Ile Ser Ser Ser Ala Thr Met500 505 510 Pro Thr Val Gly Thr Val Asn Val Thr Leu Thr Ile Ala Ala LeuAla 515 520 525 Leu Arg Met Ser Asp Thr Leu Lys Lys Glu Val 530 535 4 27PRT Burkhorderia cepacia 4 Val Arg Lys Ser Thr Leu Thr Phe Leu Ile AlaGly Cys Leu Ala Leu 1 5 10 15 Pro Gly Phe Ala Arg Ala Ala Asp Ala AlaAsp 20 25 5 16 PRT Burkhorderia cepacia 5 Ala Asp Ala Ala Asp Pro AlaLeu Val Lys Arg Gly Glu Tyr Leu Ala 1 5 10 15 6 15 DNA ArtificialSequence Description of Artificial Sequence primer 6 gcggatgcgg cggat 157 15 DNA Artificial Sequence Description of Artificial Sequence primer 7cgccagatat tcgcc 15 8 18 DNA Artificial Sequence Description ofArtificial Sequence primer 8 ccggcgctgg tgaaacgc 18 9 28 DNA ArtificialSequence Description of Artificial Sequence primer 9 cccaagcttgggccgatacc gatacgca 28 10 28 DNA Artificial Sequence Description ofArtificial Sequence primer 10 gagaagcttt ccgcacggtc agacttcc 28 11 27DNA Artificial Sequence Description of Artificial Sequence primer 11catgccatgg cacacaacga caacact 27 12 31 DNA Artificial SequenceDescription of Artificial Sequence primer 12 cccaagcttg ggtcagacttccttcttcag c 31

What is claimed is:
 1. A method for producing glucose dehydrogenasecomprising the steps of culturing a microorganism belonging to the genusBurkhorderia and having glucose dehydrogenase producing ability in amedium, and collecting glucose dehydrogenase from the medium and/orcells of the microorganism.
 2. The method for producing glucosedehydrogenase according to claim 1, wherein the microorganism isBurkhorderia cepacia.
 3. The method for producing glucose dehydrogenaseaccording to claim 1 or 2, wherein the glucose dehydrogenase has thefollowing properties: (i) the enzyme has an action of catalyzingdehydrogenation reaction of glucose; (ii) the enzyme consists ofsubunits showing a molecular weight of about 60 kDa and a molecularweight of about 43 kDa in SDS-polyacrylamide gel electrophoresis under areducing condition; (iii) the enzyme shows a molecular weight of about380 kDa in gel filtration chromatography using TSK Gel G3000SW (TosohCorporation); and (iv) the enzyme shows an optimal reaction temperaturearound 45° C. (Tris-HCl buffer, pH 8.0).
 4. The method for producingglucose dehydrogenase according to claim 3, wherein the subunit showinga molecular weight of about 43 kDa is an electron-transferring protein.5. The method for producing glucose dehydrogenase according to claim 4,wherein the electron-transferring protein is cytochrome C.
 6. A glucosedehydrogenase, which can be produced by a microorganism belonging to thegenus Burkhorderia.
 7. The glucose dehydrogenase according to claim 6,wherein the microorganism is Burkhorderia cepacia.
 8. The glucosedehydrogenase according to claim 6 or 7, wherein the glucosedehydrogenase has the following properties: (i) the enzyme has an actionof catalyzing dehydrogenation reaction of glucose; (ii) the enzymeconsists of subunits showing a molecular weight of about 60 kDa and amolecular weight of about 43 kDa in SDS-polyacrylamide gelelectrophoresis under a reducing condition; (iii) the enzyme shows amolecular weight of about 380 kDa in gel filtration chromatography usingTSK Gel G3000SW (Tosoh Corporation); and (iv) the enzyme shows anoptimal reaction temperature around 45° C. (Tris-HCl buffer, pH 8.0). 9.The glucose dehydrogenase according to claim 8, wherein the subunitshowing a molecular weight of about 43 kDa is an electron-transferringprotein.
 10. The glucose dehydrogenase according to claim 9, wherein theelectron-transferring protein is cytochrome C.
 11. The glucosedehydrogenase according to any one of claims 8 to 10, wherein thesubunit showing a molecular weight of about 60 kDa comprises the aminoacid sequence of the amino acid numbers 2 to 12 in SEQ ID NO:
 3. 12. Theglucose dehydrogenase according to any one of claims 8 to 11, whereinthe N-terminus of the subunit showing a molecular weight of 43 kDa hasthe amino acid sequence of SEQ ID NO:
 5. 13. The glucose dehydrogenaseaccording to claim 11, wherein the subunit showing a molecular weight ofabout 60 kDa is a protein defined in the following (A) or (B): (A) aprotein which has the amino acid sequence of SEQ ID NO: 3; (B) a proteinwhich has the amino acid sequence of SEQ ID NO: 3 includingsubstitution, deletion, insertion or addition of one or several aminoacid residues and a glucose dehydrogenase activity.
 14. The glucosedehydrogenase according to claim 6, which shows activity peaks around45° C. and around 75° C.
 15. A cytochrome C, which is a subunit of theglucose dehydrogenase according to claim 10 and has the amino acidsequence of SEQ ID NO:
 5. 16. A DNA encoding a part of the cytochrome Caccording to claim 15 and having the nucleotide sequence of SEQ ID NO:8.
 17. A DNA encoding a part of the cytochrome C according to claim 15and having the nucleotide sequence of the nucleotide numbers 2386 to2467 in the nucleotide sequence of SEQ ID NO:
 1. 18. A DNA encoding asignal peptide of the cytochrome C according to claim 15 and comprisingthe nucleotide sequence of the nucleotide numbers 2386 to 2451 in thenucleotide sequence of SEQ ID NO:
 1. 19. A peptide which is a signalpeptide of cytochrome C and has the amino acid sequence of the aminoacid numbers 1 to 22 in the amino acid sequence of SEQ ID NO:
 4. 20. Aprotein having the following properties: (i) the protein can constitutethe glucose dehydrogenase according to claim 6 as a subunit; (ii) theprotein has a glucose dehydrogenase activity; (iii) the protein shows amolecular weight of about 60 kDa in SDS-polyacrylamide gelelectrophoresis under a reducing condition; and (iv) the protein showsan optimal reaction temperature around 75° C. (Tris-HCl buffer, pH 8.0).21. The protein according to claim 20, which comprises the amino acidsequence of the amino acid numbers 2 to 12 in SEQ ID NO:
 3. 22. Theglucose dehydrogenase according to claim 21, wherein the protein is aprotein defined in the following (A) or (B): (A) a protein which has theamino acid sequence of SEQ ID NO: 3; (B) a protein which has the aminoacid sequence of SEQ ID NO: 3 including substitution, deletion,insertion or addition of one or several amino acid residues and aglucose dehydrogenase activity.
 23. A protein defined in the following(A) or (B): (A) a protein which has the amino acid sequence of SEQ IDNO: 3; (B) a protein which has the amino acid sequence of SEQ ID NO: 3including substitution, deletion, insertion or addition of one orseveral amino acid residues and a glucose dehydrogenase activity.
 24. ADNA encoding a protein defined in the following (A) or (B): (A) aprotein which has the amino acid sequence of SEQ ID NO: 3; (B) a proteinwhich has the amino acid sequence of SEQ ID NO: 3 includingsubstitution, deletion, insertion or addition of one or several aminoacid residues and a glucose dehydrogenase activity.
 25. The DNAaccording to claim 24, which is a DNA defined in the following (a) or(b): (a) a DNA which comprises the nucleotide sequence of the nucleotidenumbers 764 to 2380 in the nucleotide sequence of SEQ ID NO: 1; (b) aDNA which is hybridizable with a nucleotide sequence comprising thesequence of the nucleotide numbers 764 to 2380 in SEQ ID NO: 1 or aprobe that can be prepared from the sequence under a stringent conditionand encodes a protein having a glucose dehydrogenase activity.
 26. Arecombinant vector comprising the DNA according to claim 24 or
 25. 27.The recombinant vector according to claim 26, which comprises nucleotidesequences encoding the signal peptide according to claim 18 and aβ-subunit.
 28. A transformant transformed with the DNA according toclaim 24 or 25 or the recombinant vector according to claim 26 or 27.29. A method for producing glucose dehydrogenase comprising the steps ofculturing the transformant according to claim 28 to produce glucosedehydrogenase as an expression product of the DNA, and collecting it.30. A Burkhorderia cepacia KS1 strain (FERM BP-7306).
 31. A glucosesensor using an enzyme electrode including the glucose dehydrogenaseaccording to any one of claims 6 to 14, the protein according to any oneof claims 20 to 23, the transformant according to claim 27 or the strainaccording to claim
 30. 32. A glucose assay kit including the glucosedehydrogenase according to any one of claims 6 to 14 or the proteinaccording to any one of claims 20 to
 23. 33. A protein having the aminoacid sequence of SEQ ID NO:
 2. 34. A DNA encoding a protein having theamino acid sequence of SEQ ID NO:
 2. 35. The DNA according to claim 34,which comprises the nucleotide sequence of the nucleotide numbers 258 to761 in the nucleotide sequence of SEQ ID NO:
 1. 36. A DNA comprising theDNA according to claim 34 or 35 and the DNA according to claim 24 or 25in this order.
 37. The DNA according to claim 36, which comprises thenucleotide sequence of the nucleotide numbers 258 to 2380 in thenucleotide sequence of SEQ ID NO:
 1. 38. A recombinant vector comprisingthe DNA according to claim 36 or
 37. 39. The recombinant vectoraccording to claim 38, which comprises nucleotide sequences encoding thesignal peptide according to claim 18 and a β-subunit.
 40. A transformanttransformed with the DNA according to claim 36 or 37 or the recombinantvector according to claim 38 or
 39. 41. A method for producing glucosedehydrogenase comprising the steps of culturing the transformantaccording to claim 40 to produce glucose dehydrogenase as an expressionsubstance of the DNA according to claim 36 or 37, and collecting it.